The primary objective of the proposed research is to synthesize new compounds that can be used to control lipid-mediated membrane fusion. An interdisciplinary project is described that will expand the range of materials available for accelerating this fundamentally important process. The proposed materials will be incorporated within guest membrane vesicles as masked, nonfusogenic compounds that will become fusogenic upon exposure to acidic or oxidative environments--a triggering process that is conceptually similar to pH-induced viral protein-based membrane fusion within acidic endosomes. Preliminary experiments have guided the design of cleavable vinyl ether-PEG lipids that promote membrane fusion after triggering vinyl ether bond degradation. Synthetic methodology developed in the PI's laboratory will be used to install vinyl ether linkages of tunable lability within a family of phase-segregating, cleavable PEG lipids. These compounds will contain hydrophobic rod segments that are masked on one end by a cleavable hydrophilic vinyl ether-PEG unit and anchored to the membrane on the other via a phase-segregating, hydrogen-bonded hydrophobic block. Mean- field single chain theory will be used to guide the design of PEG lipids that will remain dispersed prior to activation, but form a thermodynamically stable phase-separated state after triggering has occurred. Molecular dynamics simulations will be used to generate initial inputs for the proposed mean-field calculations. This fusogen library will then be tested for their ability to promote membrane fusion in model membrane systems upon PEG cleavage and insertion of the unmasked hydrophobic rod domains into apposed bilayers. HPLC analysis and fluorescence-based assays will be used to monitor the rates of PEG lipid cleavage, membrane lipid mixing, and vesicle contents mixing under acidic or oxidative triggering conditions. Physical characterization of the membrane structures, before and after triggering, will also be performed using 31P NMR, monolayer film balance, C-TEM/C-SEM, and DSC. The most efficient fusogens will be assayed, using flow cytometry and laser confocal microscopy techniques, for their ability to effect cytoplasmic release of plasmid DNA cargo in cells targeted to internalize these carriers via receptor mediated endocytosis. PUBLIC HEALTH RELEVANCE: The primary objective of the proposed research is to synthesize new compounds that can be used to control lipid-mediated membrane fusion. An interdisciplinary project is described that will expand the range of materials available for accelerating this fundamentally important process. The proposed materials will be incorporated within guest membrane vesicles as masked, nonfusogenic compounds that will become fusogenic upon exposure to acidic or oxidative environments--a triggering process that is conceptually similar to pH-induced viral protein-based membrane fusion within acidic endosomes. The most efficient fusogens will be assayed, using flow cytometry and laser confocal microscopy techniques, for their ability to effect cytoplasmic release of plasmid DNA cargo in cells targeted to internalize these carriers via receptor mediated endocytosis.